【关键词】 melanocortin
department of biomedical sciences (d.g., c.m., s.l., c.b., s.g.), section of pharmacology, department of diagnostic services (m.-m.c., a. bertolini), section of clinical pharmacology
department of anatomy and histology (d.z.), university of modena and reggio emilia, 41100 modena, italy
department of clinical and experimental medicine and pharmacology (d.a., l.m., a. bitto, h.m., f.s.), section of pharmacology, university of messina, 98100 messina, italy
departments of human pathology (a.r.b.) and experimental medicine (r.p., m.s., d.n.), university of pavia, 27100 pavia, italy
department of neuroscience (h.b.s.), university of uppsala, 75124 uppsala, sweden
abstract
ischemic stroke is one of the main causes of death and disability. we investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. a 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe dna damage and delayed neuronal death, in the hippocampus. treatment with nanomolar doses of the melanocortin analog [nle4, d-phe7] -msh [which activates the melanocortin receptor subtypes (mc) mainly expressed in central nervous system, namely mc3 and mc4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. the selective mc3 receptor agonist 2-msh had no protective effects. pharmacological blockade of mc4 receptors prevented the neuroprotective effects of [nle4, d-phe7] -msh and worsened some ischemia outcomes. together, our findings suggest that mc4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.
introduction
transient or prolonged severe decrease in cerebral blood flow may eventually lead to delayed neuronal death (1), and ischemic stroke is one of the main causes of death and the leading cause of adult disability for impairment in several functions, including learning and memory (2). within minutes to days after a cerebrovascular accident, several pathological pathways are triggered, which may potentially damage brain cells. this is caused by several mechanisms, including excitotoxicity (due to marked release of glutamate and aspartate), inflammatory response, and apoptosis (1, 3).
several melanocortins, which are endogenous peptides of the acth/msh group and include acth-(124), -msh, shorter fragments, and synthetic analogs such as [nle4, d-phe7] -msh (ndp-msh), have a life-saving effect in animal and human conditions of circulatory shock (4, 5, 6), as well as in other severe hypoxic conditions, including prolonged respiratory arrest (7) and myocardial ischemia (8). this life-saving effect is associated with blunted nuclear factor b (nf-b) activation (9) and a marked reduction in the levels of tnf- and free radicals and intercellular adhesion molecule expression by vascular endothelium (5, 9, 10, 11). this is in agreement with the notion that melanocortins have a peculiar antiinflammatory activity (12, 13, 14). moreover, it has been shown that preventive or early-initiated treatment with -msh increases brain stem auditory-evoked potentials in dogs subjected to transient basilar artery occlusion (15), decreases cerebrocortical tnf- and il-1 expression in mice subjected to transient middle cerebral artery/carotid artery occlusion (16), and decreases local and circulating tnf- in lipopolysaccharide-induced brain inflammation in mice (17).
here we provide the first clear evidence that melanocortins cause a strong protection, with a broad therapeutic treatment window, against inflammatory, apoptotic (including dna damage), and histopathological and behavioral consequences of brain ischemia by activating central nervous system (cns) melanocortin 4 (mc4) receptors.
materials and methods
transient global brain ischemia in gerbils
for these studies, we used male mongolian gerbils (charles river laboratories, calco, lecco, italy) weighing 7080 g. housing conditions and experimental procedures were in strict accordance with the european community regulations of the use and care of animals for scientific purposes (european economic community council 89/609; italian d.l. 22-1-92 no. 116) and were approved by the committee on animal health and care of modena and reggio emilia university. transient global brain ischemia was induced, under general anesthesia with chloral hydrate (400 mg/kg ip; sigma, st. louis, mo), by occluding with atraumatic clips both common carotid arteries for 10 min (18). this experimental model represents human stroke conditions due, for example, to atherosclerotic involvement of the common carotid arteries, respiratory arrest, and cardiac arrest (19, 20). rectal and cranial (left temporalis muscle) temperatures were monitored from the induction of anesthesia until death with temperature probes and were maintained close to 37 c by means of heating lamps. animals used in behavioral studies were allowed to recover for 4 d before starting testing. sham ischemic gerbils received the same surgical procedure except that the carotid arteries were not occluded.
drugs and treatment schedules
ndp-msh (sigma), 2-msh (kindly provided by prof. paolo grieco, department of pharmaceutical and toxicological chemistry, university of naples federico ii, naples, italy), and hs024 (neosystem, strasbourg, france) were dissolved in saline (1 ml/kg) and administered ip. control animals (ischemic or sham ischemic) received equal volume of saline by the same route. for behavioral studies, ndp-msh, 2-msh, or saline was administered every 12 h (for 11 d) starting 2 min before carotid occlusion; in two other groups of animals, ndp-msh treatment started 3 or 9 h after the ischemic episode. for the assay of mapk activity and cytokine expression, gerbils were treated with a single dose of ndp-msh, 2-msh, or saline 2 min before carotid occlusion (for the assay of mapk activity: in some animals, ndp-msh was given 3 or 9 h after the ischemic episode). for the assay of caspase-3 activity, gerbils were treated with two doses of ndp-msh, 2-msh, or saline: the first dose was given 2 min before carotid occlusion (in some animals, the first dose of ndp-msh was given 3 or 9 h after the ischemic episode) and the second, 12 h after the first treatment. for the assay of dna damage, treatment schedule (first dose 2 min before carotid occlusion, then treatment every 12 h) lasted 3 d. pretreatment with hs024 (cyclic msh analog, potent and highly selective mc4 receptor antagonist) (21) or saline, when done, was performed ip 20 min before each administration of ndp-msh or saline (that is, 22 min before carotid occlusion). drug doses were chosen on the basis of previous studies (8, 9, 12, 14, 21). the different treatment protocols were chosen to perform the assays of cytokines, mapk and caspase-3 activities, and dna damage at times (see below) considered of prominent expression (1, 22, 23, 24).
assessment of spatial learning and memory
we used the morris water-maze test (25, 26) in a double-blinded manner. this test measures the ability of the gerbil to learn, remember, and go to a place in space defined only by its position relative to distal extramaze cues. the apparatus consisted of a circular white pool (80 cm in diameter and 55 cm in height) filled to a depth of 15 cm with water (27 c) rendered opaque with milk powder (18). gerbils were trained to find the spatial location of a platform of clear perspex hidden by arranging for its top surface (7 cm in diameter) to be 1 cm below the water level. latency to escape onto the hidden platform was recorded. gerbils were subjected to a 5-d training sequence (to assay learning) starting 4 d after the ischemic episode, then, 2 d after the end of learning assay, to a 1-d training (to assay memory).
histology
at the end of behavioral studies, in 56 gerbils (early or delayed treated), the brains were removed and processed as previously described (8, 26). hippocampus morphology was studied on hematoxylin-eosin stained sections (7 μm thick). glial fibrillary acidic protein (gfap) and antiapoptotic activity of cells were analyzed on slides immunocytochemically treated with monoclonal anti-gfap (zymed laboratories, san francisco, ca) and monoclonal anti-bcl-2 (dako, glostrup,denmark), respectively. the slides were incubated overnight at 4 c with the antibodies, in a moist and darkened chamber. the slides were then incubated with 1:200 streptavidin biotinylated complex (dako) for 60 min and developed in diaminobenzidine (fluka, buchs, switzerland), and counterstained in harris hematoxylin. morphological analyses were performed using an axiophot photomicroscope (carl zeiss, jena, germany). histometric analyses were performed at the magnification factor on the monitor screen of x50 (length) and x800 (thickness and cell number) using an image system (vidas, carl zeiss). thickness of the pyramidal cell layer (at level of the ca1 subfield), ischemic extent [percentage of the linear size of the hippocampus ca1-ca4 subfields containing (after hematoxylin-eosin stain) neurons having red cytoplasm and pyknotic or shrunk nuclei], viable neurons in the ca1 subfield (neurons having, after hematoxylin-eosin stain, granular cytoplasm and euchromatic nucleus with large nucleoli), number of astrocytes in the ca1 subfield (cells positive to gfap), and cells positive to bcl-2 (in the ca1 subfield) were evaluated on three different slides of serial sections for each hippocampal sample. the density of neurons, astrocytes, and cells positive to bcl-2 was estimated in a 100-μm-thick band overlapping the pyramidal cell layer.
isolation of cytoplasmic proteins
at different times after the ischemic episode, whole hippocampi were dissected from gerbil brains and immediately frozen into liquid nitrogen and stored at 80 c to permit adequate preservation of the phosphorylation state. the hippocampi were used to measure mapk activity (0.5, 3.5, and 9.5 h after the ischemic episode), tnf- and il-6 amount (2 h after), caspase-3 activity (24 h after), and concentrations of bcl-2 family proteins (bcl-2, bcl-xl, and bax: 11 d after). after homogenization in lysis buffer [10 mm tris (ph 7.4); 1% sds; protease inhibitor cocktail; phosphatase inhibitors: 100 mm na3vo4, 10 mm naf, 10 mm na4p2o7] and centrifugation, the supernatant was collected and employed for protein determination using the bio-rad protein assay kit (bio-rad, richmond, va), as previously described (27).
western blot analysis of erks, c-jun n-terminal kinases, p38 mapk, tnf-, caspase-3, il-6, bcl-2, bcl-xl, and bax
as previously described (27), cytoplasmic proteins (40 μg for each sample) were denatured, electrophoretically separated, and transferred onto nitrocellulose membranes. staining of the blots with ponceau’s solution showed that total protein amount was equal in each lane. the blots were then blocked and incubated overnight at 4 c with a primary antibody for phosphorylated erks, c-jun n-terminal kinases (jnks), and p38 mapk (cell signaling, charlottesville, va); tnf-, il-6, caspase-3, and bax (chemicon international, temecula, ca); and bcl-2 and bcl-xl (biovision, mountain view, ca). the day after, the membranes were incubated with a specific secondary antibody peroxidase conjugated (pierce, rockford, il) for 1 h at room temperature. the membranes were analyzed by the enhanced chemiluminescence system according to the protocol of the manufacturer (amersham, buckinghamshire, uk). the protein signals were quantified by scanning densitometry using a bioimage analysis system (bio-profil, celbio, italy). tnf-, il-6, bcl-2, bcl-xl, and bax values, as well as the activities of erks, jnks, p38 mapk, and caspase-3, were expressed as arbitrary units (27). the change was calculated vs. the respective sham value.
dna damage
we measured dna single-strand breaks using the comet assay, as previously described (28, 29), and dna fragmentation by means of the alkaline halo/diffusion assay (30). seventy-two hours after the ischemic episode (or sham ischemia), whole hippocampi were dissected from gerbil brains, and then the cells were lysed to obtain nucleoids. in the comet assay, after electrophoresis and staining, nucleoids were visually detected using a fluorescence microscope (olympus, tokyo, japan). one hundred nucleoids (comets) randomly selected on each of two slides/gerbil were scored and classified according to the relative intensity of fluorescence in the tail and given an arbitrary value from 0 (undamaged nucleus) to 4 (severely damaged nucleus), and then the total score was divided in two; therefore, the score in each gerbil ranged from 0400. in the alkaline halo/diffusion assay, after incubation in an alkaline hypotonic buffer and staining, the images of nucleoids were detected by means of a digital camera operating on a fluorescence microscope and analyzed on a computer by using the public domain national institutes of health image program (http://rsb.info.nih.gov/nih-image). in all ischemic gerbils, the level of dna fragmentation was quantified, as percentage of increase of nuclear diameter compared with that of sham ischemic animals, from 25 randomly selected cells/gerbil.
statistical analysis
the values obtained in the comet assay were analyzed using kruskal-wallis test followed by mann-whitney u test. mapk activities in gerbils treated 3 and 9 h after the ischemic episode were compared with those of the corresponding controls by means of student’s t test. all other data were analyzed by means of anova followed by student-newman-keuls test.
results
ndp-msh improves learning and memory performance after brain ischemia
the hippocampus, particularly the ca1 subfield, is an area of the brain that plays a critical role in learning and memory. a brief, transient period of global cerebral ischemia causes selective loss of ca1 pyramidal cells 23 d after the ischemic episode. in the mongolian gerbil, this phenomenon, termed delayed neuronal death, represents a useful model for exploring the mechanisms of neuronal death consequent to cerebral ischemia and the effects on spatial learning and memory, as well as for evaluating neuroprotective drugs in ischemic stroke (18, 31, 32, 33). therefore, we studied the ability of gerbils subjected to a 10-min period of global cerebral ischemia to learn, remember, and go to a place in space defined only by its position relative to distal extramaze cues (25, 26). in control gerbils (treated with saline), such a period of ischemia caused a significant impairment (as compared with sham ischemic) in place finding both during the first training session (assay of learning) and during the second session (assay of memory) (fig. 1, a and b). on the other hand, in gerbils treated ip (starting immediately before or 3 or 9 h after the ischemic episode) with the melanocortin ndp-msh, there was a dose-related improvement in learning and memory performance compared with ischemic control animals (fig. 1, a and b). interestingly, at the highest dose, ndp-msh accelerated learning compared with sham ischemic gerbils (fig. 1, a and c). melanocortins are known to reach the cns after systemic injection (34, 35) and much experimental evidence points in this direction (12, 13, 14, 36). five melanocortin receptor subtypes (mc1mc5) are known so far, and mc3 and mc4 are the predominant subtypes expressed in the cns (12, 13, 14, 16, 36). therefore, we investigated the role of these receptors. the protective effect of the maximally effective dose of ndp-msh [which activates mc1, mc3, mc4, and mc5 receptor subtypes (12)] on the behavioral performance of gerbils was completely prevented by a pretreatment with the selective mc4 receptor antagonist hs024 (21) (fig. 1, c and d). in these animals, as well as in those treated only with the mc4 receptor antagonist hs024, there was a worsening of memory, as compared with ischemic gerbils treated with saline. moreover, the selective mc3 receptor agonist 2-msh (12) (treatment equimolar to ndp-msh) had no protective effect (fig. 1, c and d).
ndp-msh inhibits mapk activation and proinflammatory cytokine overexpression
some members of mapks, such as jnks, p38 mapk, and erks, are early activated after transient forebrain ischemia in the gerbil hippocampus (24). the activation of some members of mapks has been implicated in the transcription of several genes involved in inflammatory processes, including genes encoding for proinflammatory cytokines (37). indeed, cytokines such as tnf- and il-6 increase early (within 13 h) in the hippocampus after brain ischemia (1, 38). in our study, immunoblot analysis showed that the activities of jnks, p38 mapk, and erks in whole hippocampus were increased in saline-treated gerbils subjected to a 10-min period of global brain ischemia, as detected 0.5, 3.5, and 9.5 h after reperfusion (40, 30, and 30 min after treatment, respectively) (fig. 2, ac). in gerbils treated with ndp-msh (also late), but not in those treated with the mc3 agonist 2-msh (at equimolar dose), there was a significant reduction of such increase (more markedly in jnks and p38 mapk); this protective effect of ndp-msh was counteracted by gerbil pretreatment with the mc4 receptor antagonist hs024 (fig. 2, ac). accordingly, tnf- and il-6 hippocampal levels were increased in saline-treated gerbils, as detected 2 h after the ischemic episode, and treatment with ndp-msh, but not with 2-msh, significantly prevented such increase (fig. 3, a and b). also this effect of ndp-msh was counteracted by pretreatment with the mc4 receptor antagonist hs024 (fig. 3, a and b).
ndp-msh reduces histological damage and delayed neuronal death
at the end of the second session of behavioral study, we processed the hippocampus for histology and histometry. in saline-treated gerbils subjected to transient brain ischemia, we observed loss of hippocampal neurons mostly inside ca1 subfield and partially replaced by glial cell hyperplasia (astrocytes), as indicated by gfap positivity (fig. 4, b and d); astrocytes spread from the pyramidal cell layer to the adjacent layers, oriens, and radiatum (fig. 4f). moreover, we found a great number of dead neurons showing pyknosis, nuclear dust, swollen perikaryon, cellular shrinkage, and absence of nissl substance (fig. 4g); accordingly, we detected scant expression of antiapoptotic activity (number of cells expressing the antiapoptotic protein bcl-2; fig. 4, e and h). in the hippocampus (from ca1 to ca4 subfield) of gerbils treated (early or late) with doses of ndp-msh maximally effective in improving the behavioral performance, we detected an ischemic extent quite similar to that of saline-treated ones (fig. 4a) but with a significantly larger thickness of the pyramidal cell layer in the ca1 subfield (fig. 4c). in this subfield, we also recorded a significantly reduced number of ischemic neurons and a higher number of viable neurons (also when the first treatment was delayed up to 3 or 9 h), when compared with the corresponding areas of saline-treated gerbils (fig. 4, d and g). in the ca1 subfield, astrocyte immunoreaction was quite similar to that observed in saline-treated gerbils, but without oriens and radiatum spread (fig. 4, b and f), whereas the antiapoptotic activity (number of bcl-2 positive cells) was more expressed (fig. 4, e and h). treatment with the selective mc3 agonist 2-msh (equimolar to ndp-msh), as expected, had no protective effects (fig. 4, ce). the effect of ndp-msh on thickness of the pyramidal cell layer, number of viable neurons, and bcl-2 immunoreaction was prevented by pretreatment of gerbils with the selective mc4 receptor antagonist hs024 (fig. 4, ce).
ndp-msh modulates the apoptotic process
the activation of mapks has been implicated in the apoptotic processes occurring after brain ischemia (1, 24, 39); in our experimental model of brain ischemia, ndp-msh blunted the activation of mapk members jnks, p38 mapk, and erks (fig 2, ac). the interaction between bcl-2 family members that promote (bax) and suppress (bcl-2 and bcl-xl) apoptosis determines whether cells undergo programmed death (40, 41, 42); in our conditions, ndp-msh caused overexpression of bcl-2 immunoreactivity in the cells of hippocampus pyramidal layer (fig. 4, e and h). this would indicate that melanocortins also affect apoptosis. to further investigate the role of melanocortins in the apoptotic process after transient global brain ischemia, at the end of the second session of behavioral study, we detected bcl-2, bcl-xl, and bax by means of immunoblot analysis. hippocampal levels of both bcl-2 and bcl-xl increased in ischemic saline-treated gerbils (fig. 5, a and b). in gerbils treated with ndp-msh (only early treatment was tested), but not in those treated with the selective mc3 agonist 2-msh (at equimolar dose), there was a further significant increase in bcl-2 and bcl-xl levels; this effect of ndp-msh was counteracted by pretreatment with the mc4 receptor antagonist hs024 (fig. 5, a and b). ndp-msh did not affect bcl-2 and bcl-xl levels in sham ischemic gerbils. similar high levels of bax protein were found in all experimental groups, including sham ischemic animals treated with ndp-msh (fig. 5c).
moreover, we explored the caspase pathway and the possible influence of ndp-msh. among the known 14 caspases, we chose to study the activity of the downstream executioner caspase-3 for its specific involvement in dna damage, with consequent cell death (1, 22, 23). in the hippocampi of saline-treated control gerbils, caspase-3 activity was significantly higher than that of sham ischemic animals, as detected 24 h after the ischemic episode [time of prominent expression (maintained at high levels for at least 72 h) of caspase-3 mrna in ca1 pyramidal neurons] (22) (fig. 6). in gerbils treated with ndp-msh, but not in those treated with the selective mc3 agonist 2-msh (at equimolar doses), caspase-3 activity was very reduced also when the first administration was delayed up to 3 or 9 h, and such effect of ndp-msh was again counteracted by gerbil pretreatment with the mc4 receptor antagonist hs024 (fig. 6).
the ultimate feature of apoptosis is dna fragmentation (1, 22, 23, 40). we, therefore, evaluated the degree of dna damage in the hippocampus by means of both the comet test (to assay dna single strand breaks, sign of initial lesions) (28, 29) and the alkaline halo/diffusion assay (to quantify dna fragmentation) (30). seventy-two hours after the ischemic episode, we found a significantly greater number of nuclei with long tail (high comet visual score) and with wide halo (high nuclear diameter due to radial diffusion of dna fragments) in the hippocampus of saline-treated control gerbils, when compared with those of sham ischemic animals (fig. 7, ad). in gerbils treated with ndp-msh, but not in those treated with 2-msh (at equimolar doses), dna damage was significantly lower when compared with that of control animals (fig. 7, ad). the selective mc4 receptor antagonist hs024 prevented such protective effect of ndp-msh against dna damage and even increased dna fragmentation as compared with that of ischemic gerbils treated with saline (fig. 7, a and b).
discussion
the third main cause of death in the united states and europe is ischemic stroke, and the only approved therapy for this condition is early thrombolysis (within 3 h) (43). early reperfusion with thrombolytic agents may reverse ischemic lesions, but recent investigations have shown that such reversible lesions may recur after 24 h (1). lesion recurrence may be related to occurrence of inflammation and delayed apoptotic death, which emphasizes the need for neuroprotective therapies targeting the mechanisms involved in brain cell damage (1, 3).
here we demonstrate that the melanocortin peptide ndp-msh significantly protects against impairment in learning and memory caused by transient global brain ischemia. this clear neuroprotective effect is dose dependent, occurs at nanomolar doses, and is seemingly mediated by brain melanocortin mc4 receptors. the ndp-msh-induced improvement in behavioral performance, which is greater than that of sham ischemic animals, is associated with a suppression of the inflammatory cascade, as indicated by the significant decrease in tnf- and il-6 levels in the hippocampus, as well as by the blunted activation of the mapk members jnks, p38 mapk, and erks. moreover, our data show that, despite a quite similar extent of ischemia in the hippocampus of all experimental groups, this protective effect is associated with a reduction of the morphological damage in the ca1 subfield, including a reduction of neuronal death and a larger thickness of the pyramidal cell layer. overexpression of bcl-2 and bcl-xl (but not bax) proteins is also induced by ndp-msh, as detected 11 d after ischemia (but not after sham ischemia), although an influence on bcl-2 family members, including bax, 12 d after ischemia cannot be ruled out. blunted activation of jnks, p38 mapk, and erks, as well as increased expression of bcl-2 and bcl-xl, indicate that ndp-msh also suppresses apoptosis. this notion is likewise supported by the data showing that ndp-msh causes inhibition of caspase-3 activity and decrease of dna fragmentation. it is, therefore, likely that melanocortins modulate (directly or indirectly) the apoptotic process in brain ischemia. these protective events seem to be triggered by activation of central mc4 receptors, because ndp-msh fails to protect the ischemic gerbils pretreated with the selective mc4 receptor antagonist hs024 (21), and 2-msh [selective agonist at mc3 receptors, the other mc subtype mainly expressed in the cns (12, 36)] has no protective effect.
our present data show, for the first time, the antiapoptotic effect of melanocortins in conditions of brain ischemia and give further evidence of the antiinflammatory activity of such neuropeptides (9, 12, 13, 14, 16, 17). our data confirm that melanocortins are able to reduce cerebral tnf- and il-1 levels in brain ischemia and are in agreement with the proposed important role of this mechanism in neuroprotection (16). it has been repeatedly reported that the antiinflammatory effects of melanocortins are adrenal independent, and ndp-msh does not stimulate adrenal glands, because it does not bind mc2 receptors (expressed in adrenal cortex and mediating glucocorticoid release) (12, 14, 36). in recent years, it has been also reported that melanocortins, including ndp-msh, stimulate the hypothalamic-pituitary-adrenal axis through central mc receptors (44, 45, 46). but, more recently, it has been found that -msh (at low doses) and -msh inhibit il-1-induced activation of the hypothalamic-pituitary-adrenal axis, also through central mc receptors (47). the possibility that the antiinflammatory effects of ndp-msh that we saw in this study may be an indirect consequence of glucocorticoid release from adrenal glands, therefore, cannot be completely ruled out.
although several innovative neuroprotective approaches have been shown to reduce brain lesions in animal models of stroke, clinical trials have failed to confirm these animal data so far, often because of toxic side effects and short therapeutic treatment window and because almost all studies aimed to block just one of the mechanisms leading to neuronal damage (1, 48). rather, effective neuroprotection may require rational polytherapy or identification of single agents active against more than just one mechanism of brain damage. recently, it has been suggested that among jnk isoforms (also activated owing to the excitatory amino acid glutamate receptor activation), jnk3 may be a potential target for neuroprotection therapies in stroke (49, 50). our data show for the first time that ndp-msh, an analog of endogenous melanocortins, which lack of appreciable toxicity (14), has a therapeutic treatment window of at least 9 h (figs. 1, 4, and 6) and affects several ischemia-related mechanisms of damage, including jnk3 activity (which contributes to both p46jnk and p54jnk) (49) (figs. 27). indeed, there seems to be a good correlation between behavioral and biomolecular changes; however, performing a statistical correlation between these parameters is beyond the scope of this investigation, and it could be the object of an additional study.
in brain ischemic areas, excitotoxicity directly and/or indirectly generates large amounts of radical species (1, 40). melanocortins, albeit lacking in scavenger properties, reduce free radical blood levels, including nitric oxide, in other hypoxic conditions, such as circulatory shock and myocardial ischemia (8, 10, 11). in brain ischemia, therefore, it is possible that, after signal transduction of ndp-msh, free radical-triggered activation of mapk and bcl-2 pathways be modulated (1, 51, 52), thus leading to antiinflammatory and antiapoptotic effects. an influence on nf-b activity―also involved in ischemic brain injury (53)―may also be hypothesized, in view of the sequential link between mapk and nf-b activation in other hypoxic conditions (54), and because of the fact that melanocortins blunt nf-b activation in the inflammatory response (9, 12, 14).
recently, it has been suggested that -msh-induced hypothermia might contribute to neuroprotection during global cerebral ischemia (55). we can, however, rule out a role of hypothermia for the protective effects observed in our study, because we maintained rectal and cranial temperatures of gerbils close to 37 c from the induction of anesthesia until death.
melanocortins are largely distributed in the cns, and mc4 receptors have been found in various brain areas including the hippocampus (12, 13, 14, 16, 36), and recently they have also been found outside the cns in the rat (56). it is known that melanocortins improve learning and memory in normal animals (57) and increase production of the antiinflammatory cytokine il-10 in modulating the inflammatory cascade (12, 14). on the other hand, low plasma concentrations of il-10 are associated with early worsening of neurological symptoms in patients with acute ischemic stroke (58), and -msh plasma levels are markedly decreased in patients with an unfavorable outcome after acute traumatic brain injury (14). accordingly, our data show an apparently faster learning in ndp-msh-treated ischemic animals, when compared with saline-treated sham ischemic ones, and a postischemic worsening of memory―as well as an increase in dna fragmentation―after the blockade of brain mc4 receptors, when compared with that of ischemic gerbils treated with saline. the suggestion that melanocortins might be physiologically involved in neuroprotection in conditions of brain ischemia, and that mc4 receptors might play an eminent role, is, for the moment, still speculative.
in conclusion, considering the broad time window for successful drug treatment (several hours), the activity against several ischemia-related mechanisms of damage, the high pharmacological potency (they are effective at nanomolar doses), and the lack of apparent toxicity (14), melanocortins, in particular selective agonists at mc4 receptors, in our opinion are worth evaluating in other animal models of brain ischemia. such studies might aid in further development of novel approaches to neuroprotection in ischemic stroke.
footnotes
this work was supported in part by grants from ministero dell’istruzione, dell’universita e della ricerca, roma, and fondazione cassa di risparmio di modena, modena, italy.
first published online october 27, 2005
abbreviations: cns, central nervous system; gfap, glial fibrillary acidic protein; jnk, c-jun n-terminal kinase; mc, melanocortin receptor subtype; ndp-msh, [nle4, d-phe7] -msh; nf-b, nuclear factor b.
accepted for publication october 20, 2005.
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